3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic role of MCC, deficiencies in this enzyme lead to organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each α- and β-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in a non-filamentous state at 2.4 Å resolution. Biotin is covalently bound to the biotin carboxyl carrier protein domain of α-subunits and positioned in a non-canonical pocket near the active site of neighboring β-subunit dimers. Moreover, flexibility of key residues at α-subunit interfaces and loops enables pivoting of α-subunit trimers to partly reduce the distance between α- and β-subunit active sites, required for MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and to assist drug discovery against trypanosome infections.

3-甲基巴豆酰辅酶 A 羧化酶 (MCC) 催化生物素依赖性的两步生成 3-甲基戊二酰辅酶 A，这是亮氨酸分解代谢的重要中间体。鉴于 MCC 的关键代谢作用，这种酶的缺乏会导致有机酸尿症，而其过度表达则与肿瘤的发展有关。 MCC 是一种十二聚体酶，由每个 α 和 β 亚基的六个副本组成。我们以 2.4 Å 分辨率展示了非丝状状态的布氏锥虫内源性 MCC 全酶的冷冻电镜结构。生物素与 α 亚基的生物素羧基载体蛋白结构域共价结合，并位于邻近 β 亚基二聚体活性位点附近的非规范口袋中。此外，α-亚基界面和环处关键残基的灵活性使得α-亚基三聚体旋转，部分减少MCC催化所需的α-和β-亚基活性位点之间的距离。我们的结果提供了一个结构框架来了解真核 MCC 的酶促机制并协助发现针对锥虫感染的药物。